Methylotrophic yeast (including Pichia, Hansenula, Candida, Torulopsis et al.) expression systems have been wildly used in industrial production and pharmacy for their efficient ability of expressing an exogenous polypeptide. It is characterized that these systems have promoters (AOX1 promoter, DHAS promoter, FDH promoter, MOX promoter, AOX2 promoter, ZZA1, PEX5-, PEX8-, PEX14-promoter et al.) which can be induced efficiently by methanol, and these promoters depend on methanol strictly while other carbon sources such as glucose, glycerol et al. will suppress the expression of these promoters.
With the outbreak of the oil crisis, the cost of producing simple-cell proteins with methanol increases. So many exogenous polypeptides are expressed with methylotrophic yeast expression systems for their incomparable advantages over other expression systems as follows: simple genetic manipulation, high expression of an exogenous polypeptide, with intracellular or secretory expression; the inheritance of an exogenous polypeptide is stable; as an eukaryotic expression system, having subcellular structures of eukaryotes, having the function of post-translational modification such as glycosylation, fatty acylation and protein phosphorylation.
As the usage scope increases, there are many problems occurring during the actual fermentation scale-up: (1) the promoter used in the expression needs to be induced by methanol, and special explosion-proof design is necessary in large scale industrial fermentation for methanol is toxic and inflammable; (2) methanol fermentation consumes large amount of oxygen, and it is difficult to meet the need for oxygen by just increasing air ventilation volume and raising rotation rate, so the pure oxygen is necessary, the oxygen desired in methanol metabolism is 3 to 4 times the amount of oxygen desired wherein glucose is the carbon source. The more methanol is consumed, the more pure oxygen is desired which bring about a great trouble to the actual industrialization. Furthermore, the more methanol is consumed, the more heat is produced which increases requirement of the cooling ability of the instrument; (3) methanol is a petrochemical product which is not suitable for some food additives production; (4) methanol metabolism will produce H2O2, which leads to the hydrolysis of the expressed polypeptides.
Therefore, if a method for expressing by a methanol inducible promoter without methanol so that the efficient transcription of a methanol inducible promoter will not depend on methanol and other carbon sources can be used to induce the expression of the promoter is obtained, it will be a positive significance for expressing exogenous polypeptides efficiently without methanol.